Antibodies to sulfated carbohydrates

ABSTRACT

The present invention relates to antibodies and antigen-binding portions thereof that specifically bind to chondroitin sulfate, particularly CS-A, CS-C and CS-E tetrasaccharides. The present invention also relates to methods of making anti-CS antibodies, pharmaceutical compositions comprising these antibodies and methods of using the antibodies and compositions thereof for diagnosis and treatment.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application No.60/802,413, filed May 22, 2006. The priority application is incorporatedherein by reference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED R&D

The U.S. Government has certain rights in this invention pursuant toGrant No. NS045061 awarded by the National Institutes of Health.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention relates generally to antibodies to sulfated carbohydrates,particularly chondroitin sulfate A (CS-A), chondroitin sulfate C(CS-C)and chondroitin sulfate E (CS-E). Antibodies to CS-A, CS-C and CS-E areprovided, as are methods of using such antibodies.

2. Description of the Related Art

Glycosaminoglycans have an inherent capacity to encode functionalinformation that rivals DNA, RNA and proteins. Specifically, thesepolysaccharides display diverse patterns of sulfation that are tightlyregulated in vivo. Kitagawa, H. et al., J. Biol. Chem. 272, 31377-31381(1997). Plaas, A. H. K. et al., J. Biol. Chem. 273, 12642-12649 (1998).Chondroitin sulfate (CS) glycosaminoglycans play important roles inbiological processes such as neural development, viral invasion, cancermetastasis and spinal cord injury. The three major sulfation motifsfound in vivo CS-A, CS-C and CS-E, differ only subtly in their sulfationpattern and are identical in terms of stereochemistry and sugarcomposition. Although glycosaminoglycans contribute to diversephysiological processes, an understanding of their molecular mechanismshas been hampered by the inability to access homogeneousglycosaminoglycan structures.

SUMMARY OF THE INVENTION

Isolated antibodies to chondroitin sulfate are disclosed in accordancewith some embodiments of the present invention. In some embodiments, theantibodies are able to bind specifically to a single type of chondroitinsulfate oligosaccharide, wherein the chondroitin sulfate oligosaccharideis selected from the group consisting of a chondroitin sulfate A (CS-A)oligosaccharide, a chondroitin sulfate C(CS-C) oligosaccharide, and achondroitin sulfate E (CS-E) oligosaccharide. The oligosaccharide maybe, for example, a tetrasaccharide or a disaccharide.

In some embodiments, an isolated antibody to chondroitin sulfate is anisolated humanized antibody.

Antibodies to chondroitin sulfate can be used to modulate the activityof chondroitin sulfate binding proteins. For example, methods ofmodulating TNF-α activity and midkine activity using an antibody thatbinds to chondroitin sulfate E is disclosed in accordance with otherembodiments. An antibody to chondroitin sulfate E can also be used tomodulate neuronal growth. In addition, methods of modulating brainderived neurotrophic factor (BDNF) activity using an antibody that bindsto a chondroitin sulfate selected from CS-E, CS-A, and CS-C areprovided.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the synthesis of CS tetrasaccharides of defined sulfationpattern, stereochemistry and chain length. Tetrasaccharides wereassembled from a core disaccharide building block 5 and elaborated toinstall distinct sulfation motifs. This modular, convergent approachpermits access to a variety of sulfation patterns, including threeimportant sulfation motifs found in the mammalian brain CS-E, CS-C andCS-A) and the CS-R motif, which has the same overall electrostaticcharge as CS-E and can be used to evaluate further the importance ofsulfate group orientation. The following abbreviations are used in FIG.1: TMSOTf, trimethylsilyl trifluoromethansulfonate; CH₂Cl₂,dichloromethane; HF.pyr, hydrogen fluoride-pyridine complex; Bu₃SnH,tri-n-butyltin hydride; AIBN, 2,2′-azobisisobutyronitrile; DDQ,2,3-dichloro-5,6-dicyano-1,4-benzoquinone; H₂O, water; CH₃CN,acetonitrile; SO₃.TMA, sulfur trioxide-trimethylamine complex; DMF,dimethylformamide; LiOH, lithium hydroxide; H₂O₂, hydrogen peroxide;NaOH, sodium hydroxide; MeOH, methanol; BzCN, benzoyl cyanide; pyr,pyridine; PhCH(OMe)₂, benzaldehyde dimethyl acetal; CSA,DL-10-camphorsulfonic acid; SO₃.TEA, sulfur trioxide-triethylaminecomplex; AcOH, acetic acid; TBS, t-butyldimethylsilyl; Bz, benzoyl; TCA,trichloroacetyl; Me, methyl; Ac, acetyl.

FIG. 2 shows the selectivity of CS antibodies for distinct sulfationmotifs based on binding to tetrasaccharide microarrays.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

Although several strategies have been developed, no methods tosystematically explore the role of specific sulfation sequences existedprior to the developments described herein. For instance, geneticapproaches that target a sulfotransferase gene perturb multiplesulfation patterns throughout the polysaccharide chain and cannot beused to study the impact of a single structural motif Holt, C. E. etal., Neuron 46, 169-172 (2005). Biochemical methods afford a mixture ofheterogeneously sulfated compounds of poorly defined linear sequence(Nandini, C. D. et al., J. Biol. Chem. 279, 50799-50809 (2004)), therebycomplicating efforts to relate a biological function to a specificsulfation sequence.

Methods for the assembly of well-defined chondroitin sulfateoligosaccharides using a convergent, synthetic approach are disclosed inaccordance with some embodiments of the present invention. In someembodiments, methods are provided using chemical synthesis to generateoligosaccharides representing each of the three major subclasses of CSfound in vivo: CS-A, CS-C, and CS-E.

The ability to synthesize CS led to the ability to make antibodies thatspecifically recognize a single CS subclass. Thus, antibodies tochondroitin sulfate and methods for using antibodies to chondroitinsulfate are disclosed in accordance with some embodiments. Isolatedantibodies are disclosed that bind to a chondroitin sulfate,particularly one of CS-A, CS-C and CS-E. In some embodiments theantibodies can be humanized or fully human monoclonal antibodies thatbind to chondroitin sulfate with high affinity, high potency, or both.In some embodiments, the antibodies specifically bind to regions of theCS that prevent the CS from interacting with a chondroitin sulfatebinding protein and thus can be used to modulate the activity of CSbinding proteins.

Antibodies that specifically recognize one or more epitopes on a singlesubtype chondroitin sulfate are disclosed in accordance with someembodiments. Such antibodies include but are not limited to polyclonalantibodies, monoclonal antibodies (mAbs), humanized or chimericantibodies, single chain antibodies, Fab fragments, F(ab′)₂ fragments,fragments produced by a Fab expression library, anti-idiotypic (anti-Id)antibodies, and epitope-binding fragments of any of the above.

The antibodies disclosed herein may be used, for example, in thedetection of chondroitin sulfate in a biological sample. In someembodiments, the anti-chondroitin sulfate antibodies (anti-CSantibodies) can be utilized as part of a technique whereby samples areanalyzed for changes in one or more particular types of chondroitinsulfate expression. In some embodiments, the anti-CS antibodies can beutilized as part of a diagnostic or prognostic technique wherebypatients may be tested for abnormal amounts or changes in the amounts ofone or more chondroitin sulfates.

Antibodies that recognize specific CS subtypes may also be utilized inconjunction with, for example, compound screening schemes, as describedbelow for the evaluation of the effect of test compounds on expressionand/or activity of chondroitin sulfate. Such antibodies may additionallybe used as a method for the inhibition of normal or abnormal chondroitinsulfate activity and for the inhibition of binding of chondroitinsulfate binding proteins to other proteins. Thus, such antibodies may,therefore, be utilized, for example, as part of treatment methods.

In some embodiments, methods of using antibodies to chondroitin sulfateto modulate a CS binding molecule activity such as, for example, growthfactor activity, are provided. For example an antibody to CS-E could beused to treat inflammation mediated by the CS binding protein TNFα.Other anti-CS antibodies could be used to modulate cell growth, such asneuronal growth.

In addition, embodiments of the invention include methods of using theseanti-CS antibodies as a treatment for a disease. For example, theantibodies are useful for treating neurological disorders, including,for example, Alzheimer's disease, arthritis, spinal cord injury orParkinson's disease. Embodiments of the invention include articles ofmanufacture comprising the antibodies. For example, one embodiment ofthe invention is an assay kit comprising chondroitin sulfate antibodiesthat is used to screen for diseases or disorders associated withchondroitin sulfate activity. In some embodiments, the kit includes abiomarker, allowing one to determine the effectiveness of the antibodyin a particular patient.

Embodiments of the invention also include cells for producing thedisclosed antibodies.

Definitions

Unless otherwise defined, scientific and technical terms used inconnection with the present invention shall have the meanings that arecommonly understood by those of ordinary skill in the art. Further,unless otherwise required by context, singular terms shall includepluralities and plural terms shall include the singular.

Generally, nomenclatures utilized in connection with, and techniques of,cell and tissue culture, molecular biology, and protein and oligo- orpolynucleotide chemistry and hybridization described herein are thosewell known and commonly used in the art, as described in various generaland more specific references such as those that are cited and discussedthroughout the present specification. See e.g. Singleton et al.,Dictionary of Microbiology and Molecular Biology 2^(nd) ed., J. Wiley &Sons (New York, N.Y. 1994); Sambrook et al. Molecular Cloning: ALaboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y. (1989)), which is incorporated herein by reference.Standard techniques are used for recombinant DNA, oligonucleotidesynthesis, and tissue culture and transformation (e.g., electroporation,lipofection). Enzymatic reactions and purification techniques areperformed according to manufacturer's specifications or as commonlyaccomplished in the art or as described herein. Standard techniques arealso used for chemical syntheses, chemical analyses, pharmaceuticalpreparation, formulation, and delivery, and treatment of patients exceptas may be discussed herein.

As utilized in accordance with the present disclosure, the followingterms, unless otherwise indicated, shall be understood to have thefollowing meanings:

“Monosaccharide,” as used herein, refers to a polyhydroxy alcoholcontaining either an aldehyde or a ketone group, i.e., a simple sugar.Monosaccharide includes reference to naturally occurring simple sugarsas well as simple sugars which have been chemically modified. Modifiedmonosaccharides include, but are not limited to, monosaccharides thathave increased or decreased sulfation or that have modified carboxyl,amino or hydroxyl groups.

“Polysaccharide” as used herein, refers to a linear or branched polymerof two or more monosaccharides that are linked by means of glycosidiclinkages.

“Polyanion,” as used herein, refers to a molecule that possesses a largenumber of negative charges. “Polyanionic carbohydrates,” as used herein,includes reference to carbohydrates that possess a large number ofnegative charges.

“Glycosaminoglycan,” as used herein, includes reference to apolysaccharide composed of repeating disaccharide units. Thedisaccharides contain an amino sugar (i.e., glucosamine orgalactosamine) and one other monosaccharide, which may be, for example,a uronic acid (i.e., glucuronic acid or iduronic acid) as in hyaluronicacid, heparin, heparan sulfate, chondroitin sulfate or dermatan sulfate,or galactose as in keratan sulfate. The glycosaminoglycan chain may besulfated on either moiety of the repeating disaccharide.

As used herein, “chondroitin” refers generally to chondroitin, saltsthereof such as chondroitin sulfate, esters thereof, and mixturesthereof. “Chondroitin sulfate” (CS), as used herein, refers generally toat least one unit in a chondroitin sulfate chain. A chondroitin sulfatechain is a sulfated glycosaminoglycan (GAG) composed of a chain ofalternating sugars (N-acetylgalactosamine and glucuronic acid).

“Antibodies” (Abs) and “immunoglobulins” (Igs) are glycoproteins havingthe same structural characteristics. While antibodies exhibit bindingspecificity to a specific antigen, immunoglobulins include bothantibodies and other antibody-like molecules which lack antigenspecificity. Polypeptides of the latter kind are, for example, producedat low levels by the lymph system and at increased levels by myelomas.

“Native antibodies and immunoglobulins” are usually heterotetramericglycoproteins of about 150,000 daltons, composed of two identical light(L) chains and two identical heavy (H) chains. Each light chain islinked to a heavy chain by one covalent disulfide bond, while the numberof disulfide linkages varies between the heavy chains of differentimmunoglobulin isotypes. Each heavy and light chain also has regularlyspaced intrachain disulfide bridges. Each heavy chain has at one end avariable domain (VH) followed by a number of constant domains. Eachlight chain has a variable domain at one end (VL) and a constant domainat its other end; the constant domain of the light chain is aligned withthe first constant domain of the heavy chain, and the light chainvariable domain is aligned with the variable domain of the heavy chain.Particular amino acid residues are believed to form an interface betweenthe light- and heavy-chain variable domains (Chothia et al. J. Mol.Biol. 186:651 (1985); Novotny and Haber, Proc. Natl. Acad. Sci. U.S.A.82:4592 (1985); Chothia et al., Nature 342:877-883 (1989)).

The term “antibody” herein is used in the broadest sense andspecifically covers intact monoclonal antibodies, polyclonal antibodies,multispecific antibodies (e.g. bispecific antibodies) formed from atleast two intact antibodies, and antibody fragments including Fab andF(ab)′2, so long as they exhibit the desired biological activity. The“light chains” of antibodies (immunoglobulins) from any vertebratespecies can be assigned to one of two clearly distinct types, called κand λ, based on the amino acid sequences of their constant domains.Binding fragments can be produced by recombinant DNA techniques, or byenzymatic or chemical cleavage of intact antibodies. Binding fragmentsinclude Fab, Fab′, F(ab′)₂, Fv, and single-chain antibodies, asdescribed in more detail below. An antibody other than a “bispecific” or“bifunctional” antibody is understood to have each of its binding sitesidentical.

The term “variable” refers to the fact that certain portions of thevariable domains differ extensively in sequence among antibodies and areused in the binding and specificity of each particular antibody for itsparticular antigen. However, the variability is not evenly distributedthroughout the variable domains of antibodies. It is concentrated inthree segments called complementarity-determining regions (CDRs) orhypervariable regions both in the Ig light-chain and heavy-chainvariable domains. The more highly conserved portions of variable domainsare called the framework (FR). The variable domains of native heavy andlight chains each comprise four FR regions, largely adopting a β-sheetconfiguration, connected by three CDRs, which form loops connecting, andin some cases forming part of, the β-sheet structure. The CDRs in eachchain are held together in close proximity by the FR regions and, withthe CDRs from the other chain, contribute to the formation of theantigen-binding site of antibodies. The constant domains are notinvolved directly in binding an antibody to an antigen, but exhibitvarious effector functions, such as participation of the antibody inantibody-dependent cellular toxicity.

In some embodiments, the antibodies provided herein are neutralizing inthat they inhibit binding of chondroitin sulfate to a chondroitinsulfate binding protein, such as, for example, TNF-α. In someembodiments they inhibit binding by at least about 20%, 40%, 60% or 80%,and more usually greater than about 85% (as measured in an in vitrocompetitive binding assay).

Depending on the amino acid sequence of the constant domain of theirheavy chains, intact antibodies can be assigned to different “classes.”There are five major classes of intact antibodies: IgA, IgD, IgE, IgG,and IgM, and several of these can be further divided into “subclasses”(isotypes), e.g. IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. The heavy-chainconstant domains that correspond to the different classes of antibodiesare called α, δ, ε, γ, and μ, respectively. The subunit structures andthree-dimensional configurations of different classes of immunoglobulinsare well known.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicalexcept for possible naturally occurring mutations that can be present inminor amounts. Monoclonal antibodies are highly specific, being directedagainst a single antigenic site. Furthermore, in contrast to polyclonalantibody preparations which include different antibodies directedagainst different determinants (epitopes), each monoclonal antibody isdirected against a single determinant on the antigen. In addition totheir specificity, the monoclonal antibodies are advantageous in thatthey can be synthesized uncontaminated by other antibodies. The modifier“monoclonal” indicates the character of the antibody as being obtainedfrom a substantially homogeneous population of antibodies, and is not tobe construed as requiring production of the antibody by any particularmethod. For example, the monoclonal antibodies to be used in accordancewith the present invention can be made by the hybridoma method firstdescribed by Kohler et al., Nature, 256:495 (1975), or can be made byrecombinant DNA methods (see, e.g. U.S. Pat. No. 4,816,567).

An “isolated” antibody is one which has been identified and separatedand/or recovered from a component of its natural environment.Contaminant components of its natural environment are materials whichwould interfere with diagnostic or therapeutic uses for the antibody,and can include enzymes, hormones, and other proteinaceous ornonproteinaceous solutes. In preferred embodiments, the antibody will bepurified (1) to greater than 95% by weight of antibody as determined bythe Lowry method, and terminal or internal amino acid sequence by use ofa spinning cup sequenator, or (2) to homogeneity by SDS-PAGE underreducing or nonreducing conditions using, for example, Coomassie blueor, preferably, silver stain. Isolated antibody includes the antibody insitu within recombinant cells since at least one component of theantibody's natural environment will not be present. Ordinarily, however,isolated antibody will be prepared by at least one purification step.

A “neutralizing antibody” is an antibody molecule that is able toeliminate or significantly reduce an effector function of a targetantigen to which it binds. Accordingly, a “neutralizing” chondroitinsulfate antibody is capable of eliminating or significantly reducing aneffector function, such as, for example, binding of a chondroitinsulfate binding protein to another protein, where the binding ismediated by chondroitin sulfate. In one embodiment, a neutralizingantibody will reduce an effector function by 1-10, 10-20, 20-30, 30-50,50-70, 70-80, 80-90, 90-95, 95-99, 99-100%.

Digestion of antibodies with the enzyme papain results in two identicalantigen-binding fragments, known also as “Fab” fragments, and a “Fc”fragment, having no antigen-binding activity but having the ability tocrystallize. Digestion of antibodies with the enzyme pepsin results inthe a F(ab′)₂ fragment in which the two arms of the antibody moleculeremain linked and comprise two-antigen binding sites. The F(ab′)₂fragment has the ability to crosslink antigen.

“Fab” when used herein refers to a fragment of an antibody thatcomprises the constant domain of the light chain and the CH1 domain ofthe heavy chain.

“Fv” is the minimum antibody fragment that contains a completeantigen-recognition and binding site. In a two-chain Fv species, thisregion consists of a dimer of one heavy- and one light-chain variabledomain in tight, non-covalent association. In a single-chain Fv species,one heavy- and one light-chain variable domain can be covalently linkedby a flexible peptide linker such that the light and heavy chains canassociate in a “dimeric” structure analogous to that in a two-chain Fvspecies. It is in this configuration that the three CDRs of eachvariable domain interact to define an antigen-binding site on thesurface of the VH-VL dimer. Collectively, the six CDRs conferantigen-binding specificity to the antibody. However, even a singlevariable domain (or half of an Fv comprising only three CDRs specificfor an antigen) has the ability to recognize and bind antigen, althoughat a lower affinity than the entire binding site.

The term “hypervariable region” when used herein refers to the aminoacid residues of an antibody which are responsible for antigen-binding.The hypervariable region generally comprises amino acid residues from a“complementarity determining region” or “CDR” (e.g. residues 24-34 (L1),50-62 (L2), and 89-97 (L3) in the light chain variable domain and 31-55(H1), 50-65 (H2) and 95-102 (H3) in the heavy chain variable domain;Kabat et al., Sequences of Proteins of Immunological Interest, 5^(th)Ed. Public Health Service, National Institutes of Health, Bethesda, Md.(1991)) and/or those residues from a “hypervariable loop” (e.g. residues26-32 (L1), 50-52 (L2) and 91-96 (L3) in the light chain variable domainand 26-32 ((H1), 53-55 (H2) and 96-101 (H3) in the heavy chain variabledomain; Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)). “FrameworkRegion” or “FR” residues are those variable domain residues other thanthe hypervariable region residues as herein defined.

The term “complementarity determining regions” or “CDRs” when usedherein refers to parts of immunological receptors that make contact witha specific ligand and determine its specificity. The CDRs ofimmunological receptors are the most variable part of the receptorprotein, giving receptors their diversity, and are carried on six loopsat the distal end of the receptor's variable domains, three loops comingfrom each of the two variable domains of the receptor.

The term “epitope” is used to refer to binding sites for antibodies onantigens. Epitopic determinants usually consist of chemically activesurface groupings of molecules such as amino acids or sugar side chainsand usually have specific three dimensional structural characteristics,as well as specific charge characteristics.

An antibody is said to bind an antigen when the dissociation constant(“K_(D)”) is ≦10 μM, preferably ≦1 μM, more preferably ≦100 nM and mostpreferably ≦10 nM. An increased or greater dissociation constant meansthat there is less affinity between the epitope and the antibody. Inother words, that the antibody and the epitope are less favorable tobind or stay bound together. A decreased or lower equilibrium constantmeans that there is a higher affinity between the epitope and theantibody. In other words, it is more likely that the antibody and theepitope will bind or stay bound together. An antibody with a K_(D) of“no more than” a certain amount means that the antibody will bind to theepitope with the given K_(D), or more strongly (or tightly).

While K_(D) describes the binding characteristics of an epitope and anantibody, “potency” describes the effectiveness of the antibody itselffor a function of the antibody. A relatively low K_(D) does notautomatically mean a high potency. Thus, antibodies can have arelatively low K_(D) and a high potency (e.g. they bind well and alterthe function strongly), a relatively high K_(D) and a high potency (e.g.they don't bind well but have a strong impact on function), a relativelylow K_(D) and a low potency (e.g. they bind well, but not in a mannereffective to alter a particular function) or a relatively high K_(D) anda low potency (e.g. they simply do not bind to the target well). In oneembodiment, high potency means that there is a high level of inhibitionwith a low concentration of antibody. In one embodiment, an antibody ispotent or has a high potency when its IC₅₀ is a small value, forexample, 130-110, 110-90, 90-60, 60-30, 30-25, 25-20, 20-15, or less pM.

“Substantially,” unless otherwise specified in conjunction with anotherterm, means that the value can vary within the amount that isattributable to errors in measurement that can occur during the creationor practice of the embodiments. “Significant” means that the value canvary as long as it is sufficient to allow the claimed invention tofunction for its intended use.

The term “selectively binds” in reference to an antibody does not meanthat the antibody only binds to a single substance. Rather, it denotesthat the K_(D) of the antibody to a first substance is less than theK_(D) of the antibody to a second substance. Antibodies that exclusivelybind to an epitope only bind to that single epitope.

The term “and/or” denotes 1) including all of the relevant options, 2)including only one (or a subset) of a number of alternative options, 3)including both of the previous descriptions 1) or 2, and 4) includingonly one of the previous descriptions (1) or 2)).

The term “treat” or “treatment” refer to both therapeutic treatment andprophylactic or preventative measures, wherein the object is to preventor slow down (lessen) an undesired physiological change or disorder,such as the development or spread of cancer or asthma.

The term “patient” includes human and veterinary subjects.

“Administer,” for purposes of treatment, means to deliver to a patient.For example and without limitation, such delivery can be intravenous,intraperitoneal, by inhalation, intramuscular, subcutaneous, oral,topical, transdermal, or surgical.

“Therapeutically effective amount,” for purposes of treatment, means anamount such that an observable change in the patient's condition and/orsymptoms could result from its administration, either alone or incombination with other treatment. As discussed herein, and as will beappreciated by one of skill in the art, there are a variety of ways inwhich an effective amount can be determined. For example, an effectiveamount can be an amount required to reduce the amount of a biomarker byany significant amount, including, for example, 0-1, 1-5, 5-10, 10-20,20-30, 30-40, 40-50, 505-60, 60-70, 70-80, 80-90, 90-95, 95-99, 99-100%of a reduction in the biomarker.

An “chondroitin sulfate related disorder” is any disease, disorder, orsimilar such term in which chondroitin sulfate regulates, influences orplays some role in the disease, optionally including the symptoms of thedisease. For example, chondroitin sulfate may play a role in modulatingthe binding of a chondroitin sulfate binding protein to another protein,where the binding is associated with the disease or disorder. Examplesinclude neurological disorders, including Parkinson's Disease,Alzheimer's Disease, Huntington's Disease, arthritis and trauma, such asspinal cord injury. In some embodiments, a “chondroitin sulfatedependent disorder” is any of the above that can be directly influencedby the administration of an antibody to chondroitin sulfate. Forexample, the disorder may be directly the result of excessive amounts ofchondroitin sulfate or the activity of a CS binding protein. In someembodiments, a chondroitin sulfate antibody treatable disorder is any ofthe above that can be effectively treated by the addition of one of thepresently disclosed antibodies.

Altering “chondroitin sulfate related activity” can include treating anyof the above disorders with an antibody; it can also include other,nontherapeutic or prophylactic uses of the antibody which can alter theactivity of chondroitin sulfate. In some embodiments, “chondroitinsulfate related disorder” can encompass any disorder in which anelevated level of chondroitin sulfate is present in the patient. In someembodiments, “chondroitin sulfate related disorder” can encompass anydisorder that has a phenotype that is characteristic of chondroitinsulfate. Phenotypes that are characteristic of a patient with achondroitin sulfate related disorder can be determined and observed byadministering an amount of chondroitin sulfate to a patient to inducevarious phenotypes. The amount of chondroitin sulfate administered canvary and can be routinely determined by one of skill in the art.

A “pharmaceutically acceptable vehicle,” for the purposes of treatment,is a physical embodiment that can be administered to a patient.Pharmaceutically acceptable vehicles can be, but are not limited to,pills, capsules, caplets, tablets, orally administered fluids,injectable fluids, sprays, aerosols, lozenges, neutraceuticals, creams,lotions, oils, solutions, pastes, powders, vapors, or liquids. Oneexample of a pharmaceutically acceptable vehicle is a buffered isotonicsolution, such as phosphate buffered saline (PBS).

“Neutralize,” for purposes of treatment, means to partially orcompletely suppress chemical and/or biological activity.

“Down-regulate,” for purposes of treatment, means to lower the level ofa particular target.

“Mammal” for purposes of treatment refers to any animal classified as amammal, including humans, domestic and farm animals, and zoo, sports, orpet animals, such as monkeys, dogs, horses, cats, cows, etc.

Fragments or analogs of antibodies or immunoglobulin molecules can bereadily prepared by those of ordinary skill in the art. Preferred amino-and carboxy-termini of fragments or analogs occur near boundaries offunctional domains. Structural and functional domains can be identifiedby comparison of the nucleotide and/or amino acid sequence data topublic or proprietary sequence databases. Preferably, computerizedcomparison methods are used to identify sequence motifs or predictedprotein conformation domains that occur in other proteins of knownstructure and/or function. Methods to identify protein sequences thatfold into a known three-dimensional structure are known. Bowie et al.Science 253:164 (1991).

Antibody Structure

The basic antibody structural unit is known to comprise a tetramer. Eachtetramer is composed of two identical pairs of polypeptide chains, eachpair having one “light” (about 25 kDa) and one “heavy” chain (about50-70 kDa). The amino-terminal portion of each chain includes a variableregion of about 100 to 110 or more amino acids primarily responsible forantigen recognition. The carboxy-terminal portion of each chain definesa constant region primarily responsible for effector function. Humanlight chains are classified as kappa and lambda light chains. Heavychains are classified as mu, delta, gamma, alpha, or epsilon, and definethe antibody's isotype as IgM, IgD, IgA, and IgE, respectively. Withinlight and heavy chains, the variable and constant regions are joined bya “J” region of about 12 or more amino acids, with the heavy chain alsoincluding a “D” region of about 10 more amino acids. (See generally,Fundamental Immunology Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, N.Y.(1989)), incorporated by reference in its entirety for all purposes).The variable regions of each light/heavy chain pair form the antibodybinding site.

Thus, an intact antibody has two binding sites. Except in bifunctionalor bispecific antibodies, the two binding sites are the same.

The chains all exhibit the same general structure of relativelyconserved framework regions (FR) joined by three hyper variable regions,also called complementarity determining regions or CDRs. The CDRs fromthe two chains of each pair are aligned by the framework regions,enabling binding to a specific epitope. From N-terminal to C-terminal,both light and heavy chains comprise the domains FR1, CDR1, FR2, CDR2,FR3, CDR3 and FR4. The assignment of amino acids to each domain is inaccordance with the definitions of Kabat Sequences of Proteins ofImmunological Interest (National Institutes of Health, Bethesda, Md.(1987 and 1991)), or Chothia & Lesk J. Mol. Biol. 196:901-917 (1987);Chothia et al. Nature 342:878-883 (1989).

A bispecific or bifunctional antibody is an artificial hybrid antibodyhaving two different heavy/light chain pairs and two different bindingsites. Bispecific antibodies can be produced by a variety of methodsincluding fusion of hybridomas or linking of Fab′ fragments. (See, e.g.Songsivilai & Lachmann Clin. Exp. Immunol. 79: 315-321 (1990), Kostelnyet al. J. Immunol. 148:1547-1553 (1992)). Production of bispecificantibodies can be a relatively labor intensive process compared withproduction of conventional antibodies and yields and degree of purityare generally lower for bispecific antibodies. Bispecific antibodies donot exist in the form of fragments having a single binding site (e.g.Fab, Fab′, and Fv).

Preparation of CS Tetrasaccharides

FIG. 1 show an embodiment of a chemical synthetic scheme to generateoligosaccharides representing three major subclasses of CS found invivo, CS-A, CS-C, and CS-E. Tetrasulfated molecule 1 displays the CS-Esulfation sequence, a motif enriched in the developing brain andassociated with the proteoglycans appican, syndecan-1 and -4,neuroglycan C and phosphacan. Disulfated molecules 2 and 3 represent themost abundant sulfation patterns in vivo, CS-C and CS-A, respectively.For comparison, tetrasulfated oligosaccharide 4, denoted CS-R, was alsosynthesized. CS-R possesses the same overall negative charge as 1 buthas sulfate groups installed at the C-2 and C-3 positions ofD-glucuronic acid (GlcA).

The synthetic route disclosed in FIG. 1 allows for the generation ofvarious CS sulfation motifs from a core disaccharide building block 5.Stereocontrol in the glycosylation reactions to form β-linkedoligosaccharides was achieved using α-trichloroacetimidate donorscontaining C-2 N-trichloroacetyl (TCA) or O-benzoyl (Bz) participatinggroups. An orthogonal protecting group strategy was developed to installthe specific sulfation sequences. In particular, p-methoxybenzylideneand Bz groups were used to mask positions that were exposed at latestages of the synthesis for sulfation. To elongate the carbohydratechain, a silyl ether was used to protect the C-4 position of GlcA andliberate a hydroxyl group nucleophile for reaction with a glycosylatingagent. Finally, a versatile chemical handle, the allyl moiety, wasappended to the reducing end of the oligosaccharides for convenientconjugation to proteins, small molecules and surfaces.

The core disaccharide building block was synthesized on a multi-gramscale from protected monosaccharides 6 and 7. For elongation of thecarbohydrate chain, the disaccharide was readily converted to a suitableglycosyl donor and acceptor pair (8 and 9). Silyl deprotection of 5using HF.pyridine followed by coupling to activated imidate 8 deliveredthe β-linked tetrasaccharide 10 with excellent stereoselectivity.

Radical-mediated conversion of the TCA to an N-acetyl group andoxidative cleavage of the p-methoxybenzylidene acetal afforded the keytetraol intermediate 11. Sulfation of 11 under vigorous conditionsgenerated the precursor to CS-E and under mild conditions yielded theprecursor to CS-C. The target CS-E and CS-C tetrasaccharides (1 and 2,respectively) were obtained after silyl deprotection and saponification.Synthesis of the CS-A tetrasaccharide 3 was achieved by selectivebenzoylation of the C-6 hydroxyl groups using benzoyl cyanide, followedby sulfation at the C-4 position. The remaining silyl and esterprotecting groups were removed as described previously to afford 3.Finally, tetrasulfated 4 was generated through formation of thebenzylidene acetal, which proved more stable than thep-methoxybenzylidene acetal during the sulfation reaction. Followingsaponification, the resulting free hydroxyl groups were sulfated and thedesired CS-R tetrasaccharide obtained after deprotection of theremaining protecting groups under mildly acidic conditions.Tetrasaccharides 1-4 were purified by size-exclusion chromatography andtheir structures confirmed by ¹H-NMR, proton decoupling experiments, andelectrospray ionization mass spectrometry (ESI).

Preparation of Antibodies

For the production of anti-CS antibodies, various host animals can beimmunized by injection with one or more chondroitin sulfatepolysaccharides, preferably homogeneous CS-A, CS-C or CS-E. Such hostanimals can include but are not limited to rabbits, mice, and rats, toname but a few. Various adjuvants can be used to increase theimmunological response, depending on the host species, including but notlimited to Freund's (complete and incomplete), mineral gels such asaluminum hydroxide, surface active substances such as lysolecithin,pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpethemocyanin, dinitrophenol, and potentially useful human adjuvants suchas BCG (bacille Calmette-Guerin) and Corynebacterium parvum. Polyclonalantibodies are heterogeneous populations of antibody molecules derivedfrom the sera of the immunized animals.

Monoclonal antibodies, which are homogeneous populations of antibodiesto a particular antigen, can be obtained by any technique which providesfor the production of antibody molecules by continuous cell lines inculture. These include, but are not limited to, the hybridoma techniqueof Kohler and Milstein, (1975, Nature 256:495-497; and U.S. Pat. No.4,376,110), the human B-cell hybridoma technique (Kosbor et al., 1983Immunology Today 4:72; Cole et al., 1983 Proc Natl Acad Sci USA80:2026-2030), and the EBV-hybridoma technique (Cole et al., 1985 inMonoclonal Antibodies And Cancer Therapy, Alan R. Liss, Inc., pp.77-96). Such antibodies can be of any immunoglobulin class includingIgG, IgM, IgE, IgA, IgD and any subclass thereof. The hybridomaproducing the mAb of this invention can be cultivated in vitro or invivo. Production of high titers of mAbs in vivo makes this the presentlypreferred method of production. An example of anti-chondroitin sulfateantibody production is provided in the Examples section below.

Anti-CS antibodies were prepared as described in the Examples sectionbelow. Briefly, one or more homogeneous chondroitin sulfatepolysaccharides were conjugated to bovine serum albumin (BSA) or keyholelimpet hemocyanin (KLH). The CS-protein conjugates were then dialyzed,and the protein concentrations were determined. Mice were immunized withthe CS-KLH conjugate and boosted five times over a period of 2 months.Spleen cells of the mice were fused to a myeloma cell line, andmulticlonal cell lines were then screened via ELISA analysis. Clonesspecific for the desired CS tetrasaccharide and with absorbance valuesgreater than 1.0 were kept for subsequent expansion. Single cell cloneswere then screened via ELISA, and clones specific for the desired CStetrasaccharide and with absorbance values greater than 1.0 wereanalyzed by dot blot analysis. Antibodies to CS preferably are able tobind specifically to a single subtype of CS.

The genetic material that encodes an antibody that specifically bindschondroitin sulfate can be isolated, and that material can be introducedinto a suitable expression vector and thereafter transfected into hostcells. Thus, anti-CS antibodies can be expressed in cell lines otherthan hybridoma cell lines. Sequences encoding particular antibodies canbe used for transformation of a suitable mammalian host cell, such as aCHO cell. Transformation can be by any known method for introducingpolynucleotides into a host cell, including, for example packaging thepolynucleotide in a virus (or into a viral vector) and transducing ahost cell with the virus (or vector) or by transfection procedures knownin the art, as exemplified by U.S. Pat. Nos. 4,399,216, 4,912,040,4,740,461, and 4,959,455 (which patents are hereby incorporated hereinby reference). The transformation procedure used depends upon the hostto be transformed. Methods for introducing heterologous polynucleotidesinto mammalian cells are well known in the art and includedextran-mediated transfection, calcium phosphate precipitation,polybrene mediated transfection, protoplast fusion, electroporation,encapsulation of the polynucleotide(s) in liposomes, and directmicroinjection of the DNA into nuclei.

Mammalian cell lines available as hosts for expression are well known inthe art and include many immortalized cell lines available from theAmerican Type Culture Collection (ATCC), including but not limited toChinese hamster ovary (CHO) cells, HeLa cells, baby hamster kidney (BHK)cells, monkey kidney cells (COS), human hepatocellular carcinoma cells(e.g. Hep G2), and a number of other cell lines. Cell lines ofparticular preference are selected through determining which cell lineshave high expression levels and produce antibodies with chondroitinsulfate binding properties.

The invention also includes functional equivalents of the antibodiesdescribed in this specification. Functional equivalents have bindingcharacteristics that are comparable to those of the antibodies, andinclude, for example, chimerized, humanized and single chain antibodiesas well as fragments thereof. Methods of producing such functionalequivalents are disclosed in PCT Application WO 93/21319, EuropeanPatent Application No. 239,400; PCT Application WO 89/09622; EuropeanPatent Application 338,745; and European Patent Application EP 332,424.

Functional equivalents include polypeptides with amino acid sequencessubstantially the same as the amino acid sequence of the variable orhypervariable regions of the antibodies of the invention. “Substantiallythe same” as applied to an amino acid sequence is defined herein as asequence with at least 80%, preferably at least about 90%, and morepreferably at least about 95% sequence identity to another amino acidsequence, as determined by the FASTA search method in accordance withPearson and Lipman, Proc. Natl. Acad. Sci. USA 85, 2444 2448 (1988).

In addition, techniques developed for the production of “chimericantibodies” (Morrison et al. 1984 Proc Natl Acad Sci USA 81:6851-6855;Neuberger et al. 1984 Nature 312:604-608; Takeda et al. 1985 Nature314:452-454) by splicing the genes from a mouse antibody molecule ofappropriate antigen specificity together with genes from a humanantibody molecule of appropriate biological activity can be used. Achimeric antibody is a molecule in which different portions are derivedfrom different animal species, such as those having a variable regionderived from a murine mAb and a human immunoglobulin constant region.Chimerized antibodies preferably have constant regions derivedsubstantially or exclusively from human antibody constant regions andvariable regions derived substantially or exclusively from the sequenceof the variable region from a mammal other than a human.

Humanized forms of the antibodies can be made by methods known in theart, for example by substituting the complementarity determining regionsof, for example, a mouse antibody, into a human framework domain, e.g.,see PCT Pub. No. WO92/22653. Humanized antibodies preferably haveconstant regions and variable regions other than the complementdetermining regions (CDRs) derived substantially or exclusively from thecorresponding human antibody regions and CDRs derived substantially orexclusively from a mammal other than a human.

Functional equivalents also include single-chain antibody fragments,also known as single-chain antibodies (scFvs). Single-chain antibodyfragments of the present invention are recombinant polypeptides whichbind CS epitopes. These fragments contain at least one fragment of anantibody variable heavy-chain amino acid sequence (VH) tethered to atleast one fragment of an antibody variable light-chain sequence (VL)with or without one or more interconnecting linkers. Such a linker maybe a short, flexible peptide selected to assure that the properthree-dimensional folding of the (VL) and (VH) domains occurs once theyare linked so as to maintain the target molecule binding-specificity ofthe whole antibody from which the single-chain antibody fragment isderived. Generally, the carboxyl terminus of the (VL) or (VH) sequencemay be covalently linked by such a peptide linker to the amino acidterminus of a complementary (VL) and (VH) sequence. Single-chainantibody fragments may be generated by molecular cloning, antibody phagedisplay library or similar techniques. These proteins may be producedeither in eukaryotic cells or prokaryotic cells, including bacteria.

Single-chain antibody fragments contain amino acid sequences having atleast one of the variable or complementarity determining regions (CDR's)of the whole antibodies described in this specification, but are lackingsome or all of the constant domains of those antibodies. These constantdomains are not necessary for antigen binding, but constitute a majorportion of the structure of whole antibodies. Single-chain antibodyfragments may therefore overcome some of the problems associated withthe use of antibodies containing a part or all of a constant domain. Forexample, single-chain antibody fragments tend to be free of undesiredinteractions between biological molecules and the heavy-chain constantregion, or other unwanted biological activity. Additionally,single-chain antibody fragments are considerably smaller than wholeantibodies and may therefore have greater capillary permeability thanwhole antibodies, allowing single-chain antibody fragments to localizeand bind to target antigen-binding sites more efficiently. Also,antibody fragments can be produced on a relatively large scale inprokaryotic cells, thus facilitating their production. Furthermore, therelatively small size of single-chain antibody fragments makes them lesslikely to provoke an immune response in a recipient than wholeantibodies.

Functional equivalents further include fragments of antibodies that havethe same, or comparable binding characteristics to those of the wholeantibody. Antibody fragments which recognize specific epitopes can begenerated by known techniques. For example, such fragments include butare not limited to: the F(ab′)₂ fragments which can be produced bypepsin digestion of the antibody molecule and the Fab fragments whichcan be generated by reducing the disulfide bridges of the F(ab′)₂fragments. Alternatively, Fab expression libraries can be constructed(Huse et al., 1989 Science 246:1275-1281) to allow rapid and easyidentification of monoclonal Fab fragments with the desired specificity.In some embodiments, the antibody fragments contain all six complementdetermining regions of the whole antibody, although fragments containingfewer than all of such regions, such as three, four or five CDRs, arealso functional. Further, the functional equivalents may be or maycombine members of any one of the following immunoglobulin classes: IgG,IgM, IgA, IgD, or IgE, and the subclasses thereof.

Techniques described for the production of single chain antibodies (U.S.Pat. No. 4,946,778; Bird 1988 Science 242:423-426; Huston et al. 1988Proc Natl Acad Sci USA 85:5879-5883; and Ward et al. 1989 Nature334:544-546) can be adapted to produce single chain antibodies againstchondroitin sulfate gene products. Single chain antibodies can be formedby linking the heavy and light chain fragments of the Fv region via anamino acid bridge, resulting in a single chain polypeptide.

Human Antibodies and Humanization of Antibodies

Human antibodies avoid some of the problems associated with antibodiesthat possess murine or rat variable and/or constant regions. Thepresence of such murine or rat derived proteins can lead to the rapidclearance of the antibodies or can lead to the generation of an immuneresponse against the antibody by a patient. In order to avoid theutilization of murine or rat derived antibodies, fully human antibodiescan be generated through the introduction of human antibody functioninto a rodent so that the rodent produces fully human antibodies. Unlessspecifically identified herein, “human” and “fully human” antibodies canbe used interchangeably herein. The term “fully human” can be usefulwhen distinguishing antibodies that are only partially human from thosethat are completely, or fully human.

Fully human antibodies can be made by any methods known in the art. Onemethod for generating fully human antibodies is through the use ofXENOMOUSE® strains of mice which have been engineered to contain 245 kband 190 kb-sized germline configuration fragments of the human heavychain locus and kappa light chain locus. See Green et al. NatureGenetics 7:13-21 (1994). The XENOMOUSE® strains are available fromAbgenix, Inc. (Fremont, Calif.).

In an alternative approach, others, including GenPharm International,Inc., have utilized a “minilocus” approach. In the minilocus approach,an exogenous Ig locus is mimicked through the inclusion of pieces(individual genes) from the Ig locus. Thus, one or more V_(H) genes, oneor more D_(H) genes, one or more J_(H) genes, a mu constant region, anda second constant region (preferably a gamma constant region) are formedinto a construct for insertion into an animal.

Diagnostic Applications

The anti-CS antibodies can be employed in any known assay method, suchas competitive binding assays, direct and indirect sandwich assays, andimmunoprecipitation assays (Zola, Monoclonal Antibodies: A Manual ofTechniques, pp. 147-158 (CRC Press, Inc., 1987)). In some embodiments,the anti-CS antibodies can be used to detect CS in a biological samplein vitro or in vivo. In some embodiments, the anti-CS antibodies can beused in a conventional immunoassay, including, without limitation, anELISA, an RIA, FACS, tissue immunohistochemistry, Western blot orimmunoprecipitation. In some embodiments, the anti-CS antibodies of theinvention may be used to detect CS from humans. In some embodiments, theanti-CS antibodies can be used to detect CS from Old World primates suchas cynomologous and rhesus monkeys, chimpanzees and apes.

For diagnostic applications, the anti-CS antibodies can be labeled witha detectable moiety. The detectable moiety can be any one which iscapable of producing, either directly or indirectly, a detectablesignal. Suitable labels for the antibody or secondary can includevarious enzymes, prosthetic groups, fluorescent materials, luminescentmaterials, magnetic agents and radioactive materials. Examples ofsuitable enzymes include horseradish peroxidase, alkaline phosphatase,β-galactosidase, or acetylcholinesterase; examples of suitableprosthetic group complexes include streptavidin/biotin andavidin/biotin; examples of suitable fluorescent materials includeluciferin, umbelliferone, fluorescein, fluorescein isothiocyanate,rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride orphycoerythrin; an example of a luminescent material includes luminol; anexample of a magnetic agent includes gadolinium; and examples ofsuitable radioactive material include ¹²⁵I, ¹³¹I, ³⁵S, ¹⁴C, ³²P, or ³H.

Any method known in the art for conjugating the antibody to thedetectable moiety may be employed, including those methods described byHunter, et al., Nature 144:945 (1962); David, et al., Biochemistry13:1014 (1974); Pain, et al., J. Immunol. Meth. 40:219 (1981); andNygren, J. Histochem. and Cytochem. 30:407 (1982).

Methods for detecting anti-CS in a biological sample are disclosed inaccordance with some embodiments of the present invention. Such methodscan comprise contacting a biological sample with an anti-CS antibody ofthe invention and detecting the bound antibody bound to anti-CS, todetect the CS in the biological sample. In some embodiments, the anti-CSantibody can be directly labeled with a detectable label. In anotherembodiment, the anti-CS antibody (the first antibody) can be unlabeledand a second antibody or other molecule that can bind the anti-CSantibody is labeled. As is well known to one of skill in the art, asecond antibody is chosen that is able to specifically bind the specificspecies and class of the first antibody. For example, if the anti-CSantibody is a human IgG, then the secondary antibody may be ananti-human-IgG. Other molecules that can bind to antibodies include,without limitation, Protein A and Protein G, both of which are availablecommercially, e.g., from Pierce Chemical Co.

In some embodiments, CS can be assayed in a biological sample by acompetition immunoassay utilizing CS standards labeled with a detectablesubstance and an unlabeled anti-CS antibody. In this assay, thebiological sample, the labeled CS standards and the anti-CS antibody arecombined and the amount of labeled CS standard bound to the unlabeledantibody is determined. The amount of CS in the biological sample isinversely proportional to the amount of labeled CS standard bound to theanti-CS antibody.

One may use the immunoassays disclosed above for a number of purposes.In some embodiments, the anti-CS antibodies may be used to detect CS incells in cell culture. In some embodiments, the anti-CS antibodies maybe used to determine the level of sulfation, and/or the amount of CS onthe cell surface after treatment of the cells with various compounds.This method can be used to test compounds that may be used to activateor inhibit CS. In this method, one sample of cells is treated with atest compound for a period of time while another sample is leftuntreated. The CS level can be measured using an immunoassay.

An immunoassay for determining cell surface levels of CS typicallyincludes the steps of labeling the cell surface proteins with adetectable label, such as biotin or ¹²⁵I, immunoprecipitating theCS-bound cells with an anti-CS antibody and then detecting the labeledCS. Another preferred immunoassay for determining the localization ofCS, e.g., cell surface levels, is by using immunohistochemistry. Methodssuch as ELISA, RIA, Western blot, immunohistochemistry, cell surfacelabeling of integral membrane proteins and immunoprecipitation are wellknown in the art. See, e.g., Harlow and Lane, Antibodies: A LaboratoryManual, New York: Cold Spring Harbor Press, 1990.

The anti-CS antibodies of the invention can also be used to determinethe levels of CS in a tissue or in cells derived from the tissue. Insome embodiments, the tissue can be a diseased or injured tissue, whichcan be excised from the patient and used in an immunoassay to determine,e.g., CS levels, cell surface levels of CS or localization of CS by themethods discussed above. The method can be used to determine if a tissueexpresses CS at a high level.

The above-described diagnostic method can be used to determine the levelof expression of CS in a tissue, which may be indicative, for example,of a disease or disorder, the state of a disease or disorder, or thatthe tissue will respond well to treatment with anti-CS antibody.

The antibodies of the present invention may also be used in vivo tolocalize tissues and organs that express CS. In some embodiments, theanti-CS antibodies can be used localize CS-expressing cells. Anadvantage of some of the anti-CS antibodies of the present invention isthat they are highly specific for a particular CS, preferably a CStetrasaccharide. In some embodiments, the method can comprise the stepsof administering an anti-CS antibody or a pharmaceutical compositionthereof to a patient in need of such a diagnostic test and subjectingthe patient to imaging analysis determine the location of theCS-expressing tissues. Imaging analysis is well known in the medicalart, and includes, without limitation, x-ray analysis, magneticresonance imaging (MRI) or computed tomography (CE). In anotherembodiment of the method, a biopsy is obtained from the patient todetermine whether the tissue of interest expresses CS rather thansubjecting the patient to imaging analysis. In some embodiments, theanti-CS antibodies may be labeled with a detectable agent that can beimaged in a patient. For example, the antibody may be labeled with acontrast agent, such as barium, which can be used for x-ray analysis, ora magnetic contrast agent, such as a gadolinium chelate, which can beused for MRI or CE. Other labeling agents include, without limitation,radioisotopes, such as ⁹⁹Tc. In another embodiment, the anti-CS antibodywill be unlabeled and will be imaged by administering a second antibodyor other molecule that is detectable and that can bind the anti-CSantibody.

In some embodiments, the anti-CS antibodies can be useful as affinitypurification agents. In this process, the antibodies are immobilized ona suitable support, such a Sephadex resin or filter paper, using methodswell known in the art.

Antibody Therapeutics

Anti-CS antibodies have therapeutic value for treating symptoms andconditions related to chondroitin sulfate activity (e.g., a chondroitinsulfate related disorder). In some embodiments, the anti-CS antibodiesdisclosed herein are used in the diagnosis, prevention or treatment ofneurological disorders, including Parkinson's Disease, Alzheimer'sDisease, Huntington's Disease, arthritis and trauma, such as spinal cordinjury.

In some embodiments, the use of the antibodies in a medicament for thetreatment of a chondroitin sulfate related disorder (a disease,condition, etc., relating to chondroitin sulfate) is contemplated. Themedicament can contain a therapeutically effective amount of theantibody. In some embodiments, the amount of chondroitin sulfateantibody in the medicament is sufficient so that at least one beneficialresult is observed, e.g., a lessening of a symptom. In some embodiments,the amount that is administered removes all of the symptoms of thechondroitin sulfate related disorder. In some embodiments, the amount issufficient so that the level of a biomarker decreases in a subject afterthe medicament has been administered. In some embodiments, the amount ofthe antibody administered is about 0.001 to 1000, 0.1 to 100, 0.5 to 50,1 to 10, 1, 3, or 10 mg of antibody/kg of subject. As will beappreciated by one of skill in the art, the actual amount of theantibody can depend upon the particular disorder (e.g., asthma, is itacute or chronic), the method of administration, the frequency ofadministration, the desired result, the characteristics of the patient,and the characteristics of the antibody. As will be appreciated by oneof skill in the art, the use of the antibody in the preparation ormanufacture of a medicament can involve any of the disclosed antibodiesin any amount, sufficient to treat the particular condition it isdirected to. Any of the herein disclosed conditions, or any chondroitinsulfate related disorders, can be the condition to be treated. In someembodiments, a medicament is prepared with one of the monoclonalantibodies (mAb) selected from the group consisting of ant-CS-C antibody5D2-1D2, anti-CS-E antibody 2D11-2A10 and anti-CS-A antibody 10G9-2B5.

As will be appreciated by one of skill in the art, the nature of thedisorder can play a role in the amount, frequency, and method ofadministration. For example, in chronic disorders, relatively largeramounts, more potent antibodies, and/or more frequently administereddoses of the antibody can be required. Similarly, in acute disorders,the amount of antibody required for treatment, including prophylaxis,can be relatively less. In subjects in which sensitization is initiallyrequired prior to the challenge, lower amounts of the antibody can bebeneficial compared to the amount required for subjects that arenaturally allergic. In such chronic systems, increased amounts of theantibody, as well as increased frequency of administration can beadvantageous. The exact amount can readily be determined by one of skillin the art, in light of the present disclosure. One of skill in the artwill further appreciate other factors and how to adjust theadministration of the antibody accordingly.

If desired, the isotype of an anti-CS antibody can be switched, forexample to take advantage of a biological property of a differentisotype. For example, in some circumstances it can be desirable for thetherapeutic antibodies against chondroitin sulfate to be capable offixing complement and participating in complement-dependent cytotoxicity(CDC). There are a number of isotypes of antibodies that are capable ofthe same, including, without limitation, the following: murine IgM,murine IgG2a, murine IgG2b, murine IgG3, human IgM, human IgG1, andhuman IgG3. It will be appreciated that antibodies that are generatedneed not initially possess such an isotype but, rather, the antibody asgenerated can possess any isotype and the antibody can be isotypeswitched thereafter using conventional techniques that are well known inthe art. Such techniques include the use of direct recombinanttechniques (see e.g. U.S. Pat. No. 4,816,397), cell-cell fusiontechniques (see e.g. U.S. Pat. Nos. 5,916,771 and 6,207,418), amongothers.

In some embodiments, the anti-CS antibodies discussed herein are mouseantibodies. In some embodiments, the anti-CS antibodies discussed hereincan be human antibodies. If an antibody possessed desired binding tochondroitin sulfate, it could be readily isotype switched to generate ahuman IgM, human IgG1, or human IgG3 isotype, while still possessing thesame variable region (which defines the antibody's specificity and someof its affinity). Such molecule would then be capable of fixingcomplement and participating in CDC.

In the cell-cell fusion technique, a myeloma or other cell line isprepared that possesses a heavy chain with any desired isotype andanother myeloma or other cell line is prepared that possesses the lightchain. Such cells can, thereafter, be fused and a cell line expressingan intact antibody can be isolated.

Accordingly, as antibody candidates are generated that meet desired“structural” attributes as discussed above, they can generally beprovided with at least certain of the desired “functional” attributesthrough isotype switching.

Biologically active antibodies that bind chondroitin sulfate arepreferably used in a sterile pharmaceutical preparation or formulationto reduce the activity of chondroitin sulfate. Anti-CS antibodiespreferably possess adequate affinity to potently interfere with thebinding of chondroitin sulfate to a chondroitin sulfate binding protein.For example, the antibody can prevent interaction of CS-E with tumornecrosis factor-α (TNF-α).

When used for in vivo administration, the antibody formulation ispreferably sterile. This is readily accomplished by any method know inthe art, for example by filtration through sterile filtration membranes.The modality of antibody administration is in accord with known methods.

EXAMPLES

The following examples are by way of illustration and not by way oflimitation.

Example 1 CS Microarray Development

This example illustrates development of CS microarrays.

To create the microarrays, a general, highly efficient strategy wasdeveloped to attach synthetic oligosaccharides to the array surface. Thepreparation of CS tetrasaccharides is described above. CS moleculesdisplaying different sulfation sequences were synthesized with an allylfunctionality on the reducing end of the sugar. This group is stable tothe chemical manipulations used to synthesize the oligosaccharides, yetit can be readily functionalized for surface conjugation. Ozonolysis ofCS-A tetrasaccharide, CS-C tetrasaccharide, CS-E tetrasaccharide andCS-E disaccharide, followed by treatment with 1,2-(bisaminooxy)ethanefurnished CS oligosaccharides with a convenient aminooxy handle forcovalent attachment to aldehyde-coated glass slides. Solutions of theaminooxy oligosaccharides in 300 mM NaH₂PO₄, pH 5.0 (10 μL/well in a384-well plate) were arrayed on Hydrogel Aldehyde slides (NoAbBiodiscoveries) by using a Microgrid 11 arrayer (Biorobotics) to deliversub-nanoliter volumes at room temperature and 50% humidity.Concentrations of carbohydrates ranged from 0-500 μM. The resultingarrays were incubated in a 70% humidity chamber at room temperatureovernight and then stored in a low humidity, dust-free dessicator.Additional details for the development of the glycosaminoglycanmicroarrays are provided in the following references: Tully, S. E. etal., J. Am. Chme. Soc. (2006) 128:7740-7741; Gama, C. I. et al., NatureChemical Biology (2006) 2(9):467-473; Shipp, E. L. and Hsieh-Wilson, L.C., Chemistry & Biology (2007) 14:195-208; each of which is incorporatedherein by reference in its entirety.

Importantly, this strategy requires minimal manipulation of the sulfatedoligosaccharides, enabling their direct conjugation in two short,high-yielding steps. Moreover, the approach is compatible with standardDNA robotic printing and fluorescence scanning technology, whichrequires only minimal amounts of material and allows a large number ofmolecular interactions to be probed simultaneously.

Example 2 Chondroitin Sulfate Antibody Preparation

This example illustrates the preparation of anti-CS antibodies.

CS tetrasaccharides prepared as described above were conjugated tobovine serum albumin (BSA) or keyhole limpet hemocyanin (KLH) asfollows. Ozonolysis of the anomeric allyl group of the CS-A, -C, and -Etetrasaccharides (0.51 μmol) was followed by treatment of each compoundwith BSA (0.34 mg, 0.0051 μmol) or KLH (0.44 mg, 0.0063 μmol) andNaCNBH₃ (0.5 mg) in H₂O (pH 9.5 using K₂CO₃) for 2 d at room temperature(RT). The CS-protein conjugates were then exhaustively dialyzed against0.01 M Na₂HPO₄, 0.15 M NaCl, pH 7.4 at 4° C., and the proteinconcentrations were determined using the BCA assay (Pierce). The epitopedensity was determined by comparing the conjugated to the unconjugatedproteins using the Habeeb assay (Shi, R.-X.; Ong, C.-N.; Shen, H.-M.Oncogene 2004, 23, 7712-7721). In short, 0.1% trinitrobenzenesulfonicacid (50 μL) and 4% NaHCO₃, pH 9.5 (50 μL) were added to the proteinsolution (10 μL) in PBS (40 μL). The mixture was incubated at 40° C. for2 h, quenched with 10% SDS (50 μL). 1 M HCl (25 μL), and H₂O (500 μL),and the absorbance at 363 nm was measured. The epitope densities for theconjugates were: CS-A-BSA conjugate=14, CS-C-BSA conjugate=16, CS-E-BSAconjugate=14, CS-A-KLH conjugate=15, CS-C-KLH conjugate=15 and CS-E-KLHconjugate=14.

For the preparation of CS-A and CS-E antibodies, Balb/c mice wereimmunized with CS-A and CS-E tetrasaccharides conjugated to keyholelimpet hemocyanin (KLH) as described above. Three Balb/c female mice,4-6 weeks old, were primed and boosted at 2-week intervals for a totalof 5 intraperitoneal injections (5 mg per injection). CS-A- or CS-E-KLHconjugates were mixed with RIBI™ adjuvant (RIBI Immunochem) for thefirst two injections, and a final series of 3 boosts was performedwithout adjuvant. Bleeds were taken 1 week after each injection andmonitored by dot blot analysis. The most responsive mouse was boostedand sacrificed after three days. Spleen cells were fused with HL-1murine myeloma cells (Ventrex) using polyethylene glycol (PEG 1500,Boehringer-Mannheim). Multiclonal and monoclonal cell lines were thenscreened via ELISA analysis. A number of CS-A and CS-E antibodies werethus identified, including the CS-A antibody 10G9-2B5 and the CS-Eantibody 2D11-2A10.

For the preparation of CS-C antibodies, mice were immunized with theCS-C-KLH conjugate and boosted five times over a period of 2 months with50 μg of the conjugate at each boost. The final boost was performed 3 dbefore fusion of the spleen cells to a myeloma cell line. Multiclonalcell lines were then screened via ELISA analysis. The BSA conjugates (1μg/mL in 50 mM Na₂CO₃, pH 9.6) were added to a 384-well NUNC Maxisorpclear plate (25 μL per well), and the plate was sealed and incubated for12 h at 4° C. The wells were aspirated, washed four times with PBScontaining 0.05% Tween-20 (PBST, 75 μL/wash), and blocked for 2 h at RTwith 10% horse serum (Gibco) in PBS (75 μL). After the blocking step,the plate was washed four times with PBST, and the supernatants from theantibody producing cultures (25 μL) were added to the wells andincubated at room temperature for 2 h. Following aspiration, the wellswere washed four times with PBST and treated with a horseradishperoxidase (HRP)-conjugated goat anti-mouse antibody (Pierce; 1:10,000,25 μL/well) in blocking buffer for 1 h at RT. The wells were againaspirated, washed four times with PBST, and then developed with ABTSliquid substrate solution (Sigma; 25 μL/well, solution at RT) for 30 minat RT. Color development was monitored on a Victor plate reader(PerkinElmer) at 405 nm. Only clones specific for the CS-Ctetrasaccharide and with absorbance values greater than 1.0 were keptfor subsequent expansion. Single cell clones were then screened viaELISA as previously described, and clones specific for the CS-Ctetrasaccharide and with absorbance values greater than 1.0 wereanalyzed by dot blot analysis. A number of CS-C antibodies were thusidentified, including CS-C antibody 5D2-1D2.

Example 3 Specificity of Anti-CS Antibodies

This example illustrates specificity of CS antibodies using the CSmicroarray and dot blot analysis.

The CS microarray prepared as described in Example 1 were treated withNaBH₄ prior to use to quench unreacted aldehyde groups. The microarrayswere then incubated with monoclonal antibodies raised against CS-A, CS-Cor CS-E tetra-saccharide conjugated to keyhole limpet hemocyanin, andantibody binding was visualized using a secondary Cy3-conjugated goatanti-mouse antibody. The CS-A antibody 10G9-2B5 bound to the CS-Atetrasaccharide in a concentration-dependent manner, and strongselectivity for the CS-A motif was observed, with little detectablebinding to the CS-C or CS-E sulfation motifs, as shown in FIG. 2.Similarly, the CS-E antibody 2D11-2A10 selectively recognized the CS-Etetrasaccharide and displayed only weak binding to the CS-C motif athigh tetrasaccharide concentrations, while the CS-C antibody 5D2-1D2selectively recognized CS-C and did not display any appreciable bindingto CS-E or CS-A (FIG. 2). To examine the carbohydrate chain lengthrequired for interaction, the ability of the CS-E antibody to bind CS-Edi- and tetrasaccharides was compared. The CS-E disaccharide showedsignificantly reduced antibody binding, indicating a clear preference ofthe antibody for tetrasaccharide epitopes.

The antibody specificities obtained from the microarray were confirmedby traditional dot blot analyses. CS-A tetrasaccharide, CS-Ctetrasaccharide and CS-E tetrasaccharide were covalently attached tobovine serum albumin (BSA) by oxidation to the corresponding aldehydes,followed by reductive amination to link the carbohydrates to lysineresidues of the protein. The CS-BSA conjugates were spotted ontonitrocellulose membranes and incubated with the CS-A (10G9-2B5) or CS-E(2D11-2A10) antibody. Antibody binding was visualized bychemiluminescence using a secondary goat anti-mouse antibody conjugatedto horseradish peroxidase. Consistent with the microarray data, highlyselective binding of the antibodies to their respective sulfatedantigens was observed.

Example 4 Method of Detecting CS in a Biological Sample

CS polypeptides can be detected in a biological sample, and if anincreased or decreased level of CS is detected, the respective CS is amarker for a particular phenotype. Methods of detection are numerous,and thus, it is understood that one skilled in the art can modify thefollowing assay to fit their particular needs.

For example, antibody-sandwich ELISAs are used to detect CS in a sample,preferably a biological sample. Wells of a microtiter plate are coatedwith specific antibodies to CS, respectively, at a final concentrationof 0.2 to 10 ug/ml. The wells are blocked so that non-specific bindingof CS to their respective well is reduced.

The coated wells are then incubated with a sample containing CS.Preferably, serial dilutions of the sample should be used to validateresults. The plates are then washed to remove unbounded CS.

Next, specific anti-CS antibody-alkaline phosphatase conjugate is addedand incubated. The plates are again washed to remove unboundedconjugate.

A substrate solution is added to each well and incubated for theappropriate development time. The reaction can then be measured by amicrotiter plate reader. A standard curve is prepared using serialdilutions of a control sample, and the CS polypeptide concentration of asample can be determined using the standard curve.

Example 5 The CS-E Sulfation Motif Stimulates Neuronal Growth

This example illustrates specificity of CS-E stimulation of neuronalgrowth.

Neuritogenic activity of CS-A tetrasaccharide, CS-C tetrasaccharide andCS-E tetrasaccharide was compared using neuronal cultures. Primaryhippocampal neurons from embryonic day 18 (E 18) rats were cultured oncoverslips coated with polyornithine and CS-A tetrasaccharide, CS-Ctetrasaccharide, CS-E tetrasaccharide or CS-R tetrasaccharide. Theneurons were fixed after 48 h, immunostained with anti-tubulinantibodies, and examined by confocal fluorescence microscopy.

Notably, a specific CS sulfation pattern was required for theneuritogenic activity of CS. Whereas the CS-E tetrasaccharide stimulatedneurite outgrowth by 48.6±2.3% relative to the polyornithine control,tetrasaccharides representing other CS subclasses found in vivo, CS-Aand CS-C, had no appreciable activity. Moreover, CS-R had no effect onneurite outgrowth, despite having the same overall negative charge asCS-E. Thus, altering the precise orientation of the sulfate groups has acritical impact on the growth-promoting ability of CS.

Dopaminergic neurons from the mesencephalon of rat embryos were culturedon a substratum of each tetrasaccharide. The CS-E tetrasaccharide had asimilar activity toward both dopaminergic and hippocampal neurons,inducing the outgrowth of dopaminergic neurons by 29.6:1:6.0%. Incontrast, the CS-C, CS-A and CS-R motifs exhibited no significantneuritogenic activity. Similarly, CS-E tetrasaccharide, but not othersulfation motifs, stimulated the outgrowth of dorsal root ganglion (ORO)neurons derived from the spinal cord.

To investigate whether CS recruits specific growth factors to the cell,hippocampal neurons were treated with the CS-E tetrasaccharide in thepresence or absence of antibodies selective for midkine or BDNF. Theantibodies were expected to block the interaction of the endogenousgrowth factors with the CSE substratum and thereby abolish theneuritogenic effects. Antibodies against midkine or BDNF had no effecton neurite outgrowth in the absence of the tetrasaccharide. Importantly,addition of either antibody blocked the neurite outgrowth induced byCS-E. In contrast, a control antibody selective for FGF-1 could notabolish the growth-promoting effects of CS-E.

As further confirmation, antibodies selective for the cell surfacereceptors, protein tyrosine phosphatase zeta (PTPζ) and tyrosine kinaseB receptor (TrkB) were used. Binding of midkine and BDNF to PTPζ andTrkB, respectively, has been shown to promote neuronal outgrowth andsurvival in various systems by activating intracellular pathways such asmitogen associated protein kinase (MAPK), and phosphatidylinositol-3kinase (PI3-K) pathways. Antibodies against either PTPζ or TrkB, but notTrkA, blocked the neuritogenic activity of CS-E. In contrast, neitherantibody alone had an effect on neurite outgrowth. These resultsindicate that the CS-E sulfation motif stimulates neuronal growththrough activation of midkine-PTPζ and BDNF-TrkB signaling pathways.

Coverslip preparation, hippocampal cultures and neurite outgrowthmeasurements were performed as described in Jacquinet, J.-C. et al.,Carbohydr. Res. 314, 283-288 (1998), incorporated herein by reference inits entirety. DRG cultures were prepared from the spinal cord of E18embryos of Sprague-Dawley rats. Ganglia were dissected in Calcium andMagnesium Free-Hanks Balanced Salt Solution [CMF-HBSS (Gibco)], digestedwith 0.25% trypsin (GibeD) for 20 min at 37° C., and dissociated inculture media consisting of DMEM-F12 (Gibco), 10% horse serum (GibeD),N2 supplement (Gibco), and NGF (50 ng/mL; Gibco). DRG neurons wereplated at 100 cells/mm² on covers lips coated with poly-DL-ornithine andthe tetrasaccharides (50 or 100 μg/mL). After 24 h, neurons wereimmunostained with an anti-tubulin III antibody (Sigma, 1:500) andexamined by confocal fluorescence microscopy. Cells were imaged on aZeiss Axiovert 100M inverted confocal microscope, and images werecaptured with LSM Pascal software. Mesencephalic cells were cultured ondishes coated with polyornithine and the tetrasaccharides (16 or 50μg/mL) for 5 days, immunostained with an anti-tyrosine hydroxylaseantibody (Pel-Freeze; 1:1,000), and examined by confocal fluorescencemicroscopy. The neurite length is expressed as total length of theneurite from the perikarya, and only cells with neurites longer than onecell body diameter were counted, as per standard protocol. The length ofthe longest neurite was measured using NIH Image 1.62 software. The meanneurite lengths were compared among the different substrate conditionsby the ANOV A test followed by the Scheffe test using the statisticalanalysis program StatView (SAS Institute Inc.).

For the antibody treatments, hippocampal neurons were cultured on asubstratum of poly-DL-ornithine in the presence or absence of CS-Etetrasaccharide (500 μg/mL). After 24 h, antibodies selective formidkine (Santa Cruz; 4 μg/mL), BDNF (Santa Cruz; 1 μg/ml), FGF-1 (R & DSystems; 4 μg/mL), PTPS (Santa Cruz; 2 μg/mL), TrkB (Santa Cruz; 1μg/mL), or TrkA (Santa Cruz; 4 μg/mL) were added to the medium (500 μL).Neurons were cultured for an additional 24 h before immunostaining withan anti-tubulin antibody (Sigma; 1:500) and microscopy analysis.Relative concentrations of the CS tetrasaccharides were determined bymeasuring the uronic acid content using the carbazole reaction.

Example 6 Treatment of Spinal Cord Injury

This example illustrates the treatment of a mammal after spinal cordinjury.

A mammal suffering from spinal cord injury is identified andadministered an effective amount of a composition comprising ananti-CS-E antibody. The appropriate dosage and treatment regimen can bereadily determined by the skilled artisan based on a number of factorsincluding the nature of the anti-CS-E antibody, the route ofadministration and the mammal's injury state. Spinal cord injurytreatment efficacy is evaluated by observing delay or slowing of injuryprogression, amelioration or palliation of the injury state.

Example 7 Treatment of Inflammatory Disease

This example illustrates the treatment of an inflammatory disease usingan antibody to CS-E.

A mammal suffering from an inflammatory disease associated with TNFα isidentified and administered an effective amount of a compositioncomprising an anti-CS-E antibody. The appropriate dosage and treatmentregimen can be readily determined by the skilled artisan based on anumber of factors including the nature of the anti-CS-E antibody, theroute of administration and the mammal's injury state. Treatmentefficacy is evaluated by observing reduction in the symptoms of thedisease.

Hybridoma Deposit

Hybridomas producing each of the three antibodies discussed above(5D2-1D2, 2D11-2A10, and 10G9-2B5) are being deposited with the AmericanType Culture Collection, PO Box 1549, Manassas, Va. 20108, under theTerms of the Budapest Treaty. The Accession Numbers for the three clonesare PTA-_, and PTA-_and PTA-_, respectively.

All numbers expressing quantities of ingredients, reaction conditions,and so forth used in the specification and claims are to be understoodas being modified in all instances by the term “about.” Accordingly,unless indicated to the contrary, the numerical parameters set forth inthe specification and attached claims are approximations that can varydepending upon the desired properties sought to be obtained by thepresent invention. At the very least, and not as an attempt to limit theapplication of the doctrine of equivalents to the scope of the claims,each numerical parameter should be construed in light of the number ofsignificant digits and ordinary rounding approaches.

The invention illustratively described herein suitably can be practicedin the absence of any element or elements, limitation or limitationsthat is not specifically disclosed herein. The terms and expressionswhich have been employed are used as terms of description and not oflimitation, and there is no intention that in the use of such terms andexpressions indicates the exclusion of equivalents of the features shownand described or portions thereof. It is recognized that variousmodifications are possible within the scope of the invention disclosed.Thus, it should be understood that although the present invention hasbeen specifically disclosed by preferred embodiments and optionalfeatures, modification and variation of the concepts herein disclosedcan be resorted to by those skilled in the art, and that suchmodifications and variations are considered to be within the scope ofthis invention as defined by the disclosure.

All patents and publications are herein incorporated by reference intheir entireties to the same extent as if each individual publicationwas specifically and individually indicated to be incorporated byreference.

1. An isolated antibody which binds to a chondroitin sulfateoligosaccharide, wherein the chondroitin sulfate oligosaccharide isselected from the group consisting of a chondroitin sulfate A (CS-A)oligosaccharide, a chondroitin sulfate C(CS-C) oligosaccharide, and achondroitin sulfate E (CS-E) oligosaccharide.
 2. The isolated antibodyof claim 1, wherein the oligosaccharide is a tetrasaccharide or adisaccharide.
 3. The isolated antibody of claim 1, wherein thechondroitin sulfate is CS-A.
 4. The isolated antibody of claim 3,wherein the isolated antibody is antibody 10G9-2B5.
 5. The isolatedantibody of claim 1, wherein the chondroitin sulfate is CS-C.
 6. Theisolated antibody of claim 5, wherein the isolated antibody is antibody5D2-1D2.
 7. The isolated antibody of claim 1, wherein the chondroitinsulfate is CS-E.
 8. The isolated antibody of claim 7, wherein theisolated antibody is antibody 2D11-2A10.
 9. The isolated antibody ofclaim 1, wherein the isolated antibody is an isolated humanizedantibody.
 10. The isolated antibody of claim 1, wherein the isolatedantibody is an isolated human antibody.